Standard

The prepped plasmids from the positive clones were cut with Nco/ Xho in order to see if the 900 bp were flipping out of the pet16b backbone.

Incubation at 37°C for 40 min.

The gel showed that all picked clones were positive. Clone 17 was sent was sequencing and transformed into E.ColiBL21(DE3).

In order to check the expression of the StrepDARPidin a colony containing piGEM-028 (pet16b-StrepDARPidin) was used to inoculate 20 mL of LB-Amp. The culture incubated at 37°C until an OD of 0,7.

A ca. 1 mL preinduction probe (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL). After that the cultures were induced with 20 µL IPTG for 3h.

An induction probe (I) was taken (320 µL with an OD of 2,2; 350 µL with an OD of 2).

PI and I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and the pellet resuspended in 60 µL water and 40 µL SDS-Buffer.

The four probes were analysed on an SDS-PAGE gel with 10 µL volume per probe.

The gel showed a positive overexpression of a protein at ca. 30 kDa which fits the calculated molecular mass.

The amplification of KSII did not work: some unspecific bands were visible but not of the expected size. The amplification of flank 1 amyEfw, flank 3 lacAfw and flank 4 lacA rev was successful but yielded in a low amount of PCR product. The primers and templates for amplifying KSII, amyE rev and AmyE/yvyD were given to Florian Altegoer and were amplified by him: KSII did not work either but the flanks.

The plasmids of 13.78 were prepped at the weekend. Frozen and competent B. subtilis cells were thawed without ice. 15 µl of each plasmid were given to the cells that were incubated at 37°C for 0.5 hours. After regeneration the cells were plated on LB-Cm plates (5 ng/µl) and were incubated at 30°C for several days.

Test restriction with the isolated plasmid was performed with the corresponding enzymes (EcoRI and PstI)over night. Besides 600 ng of the already existing PCR fragments Hag-KpnI and StrepDARPidin were restricted with the same enzymes.

Concentrations:

Hag-Kpn 130 ng/µl

Strep-DARPidin 55 ng/µlafter purification

psB1C3 130 ng/µl after plasmid isolation

Restriction:(20µlreaction mix)

The bands were at a height of ca 1500 bp which is too small for the expected fragment of 1847 bp. For this reason, Florian tried the Fusion PCR as well but failed too.

The attempts were incubated at 50°C for an hour and transformed into E. Coli XL1-Blue, plated out on LB-Amp. An additional reaction was started without pMAD in a lower(1 µl) and a higher (2 µl) amount of amyE flank I. These reactions were used to do a PCR reaction with the primers piGEM034 and piGEM037 to test if the assembled fragment is present in the reaction. The expected bands should have a size of 1847 bp but the fragment visible is only ca 1300 bp big. It seems like the flank I is missing but then the fragment should not have been amplified, since the primers were chosen to include both flanks.

Information: iBB8 has a size of 1461 bp, vector 2000bp, Inserts Hag-KpnI and StrepDARPidin 1000 bp

Image of the gelextraction, as a marker the1kb gene ruler was used, 1=strepDARPidin, 2= Hag-Kpn, 3 and 4= 1C3

Concentrations:

1C3= 18 ng/µl

StrepDARPidin= 5 ng/µl

Hag-KpnI= 13 ng/µl

Ligation at RT for 90 minutes

After the ligation the whole reaction mix was transformed into chemically competent E. coli XL-1-blue cells (standard protocol). Cells were plated on LB-Cm plates and incubated at 37°C over night.

Since the Gibson assembly of the single parts without the vector (22.7) and the Fusion PCR yielded no positive results, the KSI should be amplified once again to assure usage of the correct and pure fragments for the FusionPCR or Gibson assembly.

The gel showed the expected band for the killswitch module 1 but only one band for flank 1 in the reaction with the annealing temperature of 58°C. The remaining reactions for KSI were pooled and purified with a Gel Ex kit. Since the only band for the flank 1 is visible at 58°C and not above another PCR was prepared with lower annealing temperatures but the same reaction mix as above.

All plasmids (from positive clones 13.78) were again used to transform B. subtilis PY79, since the former plates seemed to be contaminated by other organisms. E. coli DH5α was transformed with the rest of the plasmids to multiply it for later purposes.

Three colonies were grown on the plate with the Gibson assembly transformed XLIBlue cells. The control also contained three colonies. However, the colonies of the Gibson plate were tested by colony PCR with the primer surrounding the flanks of the connected KSI module, since no primer were available which target regions in the vector pMAD.

Colonies could be detected on both transformation plates, whereas no colonies grew in the ligation control plate. Therefore a colony PCR will be performed in order to analyze the transformants for the insertion of the fragments into the plasmid. 5 clones from both plates were chosen for the colony PCR and inoculated in liquid LB-Cm for plasmid isolation in the evening (in case positive clones can be detected in the colony PCR).

Colony PCR

Saved colony PCR program in the PCR cycler was used with 1 minute elongation time.

Colony PCR negative, therefore plasmids were isolated and digested with the EcoRI and PstI.

Restriction: 4µl plasmid in a total 10µl restriction mix

Restriction positive 2kb plasmid and 1kb insert), one clone each will be send for sequencing.

3 L LB medium were prepared with 1 mL ampicillin each and inoculated with some clones from the Bl21 transformation plate. The cultures were induced with 50 ml lactose solution (12,5g/ 50 mL) each and incubated at 30°C overnight.

In order to check the expression of the StrepDARPidin a colony containing piGEM-028 (pet16b-StrepDARPidin) was used to inoculate 20 mL of LB-Amp.

The fragments were all amplified and purified. This time a gradient was performed with an initial step of 50°C for 0.5 hours (similar to a Gibson assembly), since Florian tried it before and yielded better results than without the initial 50°C step.

The bands had the expected size of ca 1800 bp.

iGEM Primer in the list of last year’s team number 37 and 38 (Sample premix)

AGB0023-642= StrepDARPidinFw (Clone 1)

AGB0023-643=StrepDARPidinRv

AGB0023-644= Hag-KpnIFw (Clone 4)

AGB0023-645= Hag-KpnIRv

The 3 x 1 liter LB culture which were induced overnight with lactose were harvested and the pellets resuspended in 15 mL Buffer A. The cell suspension was cracked with the micro fluidizer. The lysate was centrifuged at 20000 rpm for 20 min and purified with a Ni-NTA Column.

The following steps were performed for the Ni-NTA Column:

• equilibration with Buffer A 10 min
• taking 40µL supernatant (load)+ 10 µL SDS-Buffer - L-Probe
• 50 mL load on column
• taking 40 µL of flow through + 10 µL SDS-buffer - FT-Probe
• first washing with 25 ml Buffer A (half the load)
• taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Probe
• equilibrate with Buffer B (output pipe off the column into glas, input into B)
• hanging pipe on column, 20 ml Evolution- E
• taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-probe
• SDS-PAGE analysis

regeneration of column:

• 10min water
• 10min EDTA
• 10min water
• 10min NiSO₄
• 10min water

Meanwhile the elution was transferred in a concentrator column which was centrifuged at 4000 rpm until the volume in the filter was 1.5 mL for injection into the gel filtration station.

The gel showed that there was not the desired StrepDARPidin loaded on the column. A SDS-PAGE probe from the pellet showed us that the pellet contained the protein.

We considered that the StrepDARPidin might be in inclusion bodies because of the Streptavidin part and the codon optimization. So we tried expression overnight with 0.1mM IPTG of an 100 mL culture at OD 0,7. We induced as well with lactose overnight as a control.

The 4 cultures were incubated at 20°C and 30°C in the shaker overnight.

piGEM-026 and -027 were used as a PCR template to amplify Hag-D2 constructs for cloning into pet24d in order to express the flagellin modification for crystallization. Fragments the size of ca. 1450 for Hag-D2-Cup and 1350 for Hag-D2-Strep were expected.

analysis:

The PCR Probes were purified and pooled with the Omega Gel Extraction Kit due to the Ezymatic reaction purification protocol. Final concentrations:

Hag-D2-Cup: 41 ng/ µL

Hag-D2-Strep: 25 ng/ µL

The PCR products from 19.12 were cut with NcoI and BamHI to get the restriction sites for cloning into pet24d Nco/BamHI cut.

Incubation overnight at room temperature.

The fragment of the fusion PCR (22.10) was separated on a 1% agarose gel, cut out and purified with a Gel Ex kit. The already cut pMAD plasmid was used to perform a Gibson assembly.

The protocol and the strains were provided by AG Bremer of the microbiology department of the Philipps Universität Marburg. All media were prepared as possible (methods) and the strains B. subtilis 168 and B. subtilis JH642 were streaked on LB-Agar and incubated at 37°C.

The cultures from the day before were spinned down at 4000 rpm for 20 min. The pellets were resuspended in 10 ml Buffer A and cracked with the micro fluidizer. The lysate was centrifuged at 20000 rpm for 20 min and purified with a Ni-NTA Beads from Qiagen according to the protocol. Probes from the pellet, supernatant and Elution wereanalysed on an SDS-PAGE gel:

The gels showed that the protein remains in the pellet no matter which attempt.

For further experiments for the isolation of the protein in inclusion bodies new expression cultures from a freshly transformed BL21(DE3) strain with piGEM-028 were used to inoculate 3 L expression culture with lactose overnight.3 L LB were prepared with 1 mL ampicillin each. The cultures were induced with 50 ml lactose solution (12,5g/ 50 mL) each and incubated at 30°C overnight.

The Nco/ Bam restricted PCR fragments from 19.13 were ligated into precut pet24d. The 20 µL attempts were transformed into E.Coli XL1-Blue after inactivation (10min at 65°C) and selected on LB- Amp plates incubated overnight at 37°C.

The control plate showed no colonies in comparison the ligation plates I and II over 15 clones. 6 Clones per plate were picked for a inoculation of minipreps for a test digest next day.

The cultures were spinned down at 4000 rpm. The pellets were resuspended in 10 ml Buffer A and centrifuged down at 4000 rpm again. The supernatant was discarded and the pellets were frozen at -80°C.

Sequencing results –positive for both sent plasmids.

In order to rescue the StrepDARPidin from the inclusion bodies we tried different protocols. The pellet from 07.09.2014 was warmed up and the cells were cracked with the microfluidizer after resuspension in 15 mL Buffer A (Lys(ate)).

The suspension was centrifuged at 20.000 x g for 15 min. The supernatant (S1) was discarded and the pellet (P1)resuspended in 5 mL 6M Guanidinium-HCL solution for solubilisation and unfolding of the StrepDARPidin.

After centrifugation at 4000 rpm for 10 min the supernatant (S2) was used for refolding. The membranous pellet (P2) was thrown away.

For refolding 100 mL Buffer A were filled into a Erlenmeyer-Flask on ice with a magnet stirrer inside on 600 rpm.

For refolding it was necessary to dilute the Guanidinium-HCL dilution very fast and softly. For that purpose rapid dilution was done by pipetting the supernatant drop by drop into the vortex of the Buffer A. After adding the whole Guanidinum-HCL/ Protein solution the suspension was observed and precipitation noticed. The suspension was centrifuged at 20.000 rpm (Pellet P3). The supernatant was loaded on a Ni-NTA Column from GE-Healthcare (L). Flow through (FT), Wash (W) in Buffer A and Elution (E) in Buffer B were kept for gel analysis. The probes lysat to Load were cooked for 5 min at 95 degrees.

From every fraction 40 µL were taken for a commassie SDS-PAGE and 10 µL 10x SDS Buffer added. The pellet probes were resuspended in 60 µL water and 40 µL SDS-Buffer.

The gel showed us that the StrepDARPidin is still in the pellet fraction but we were able to make some of it soluble as it can be seen in S2 and the load. The StrepDARPidin was thought to build tetramers with ca. 120 kDa size. The complex seems to be stabil enough to be seen on the gel above the marker which fits to the size of ca. 120 kDa- Cooking up would break the tetramer.

In order to get enough protein for a gel filtration run 4 L of BL21 with piGEM-028 were induced with lactose overnight.

The plasmids from 6 clones per Ligation plate were prepped and ca. 350 ng DNA were digested with Nco/Bam in order to check the right insertion of the 1350-1450 bp sized inserts.

Digest at 37°C for half an hour.

Upper half of the gel shows hag-d2 cup, lower half shows hag d2-strepdarp; 1kb gene ruler ladder was used. 3 clones seem to be positive for cup, but as an mixture occurred, the cloning procedure will be repeated from the PCR step on.

Template was diluted plasmid piGEM026/027

After the PCR the fragments were purified via gel extraction for further cloning steps. In the gel the cut vector pet24d was also purified.

Concentrations after clean up:

Pet24d= 20 ng/µl, D2-Cup= 208 ng/µl, D2-Strep= 180 ng/µl

After the clean up the two fragments they had to be digested with NcoI and BamHI in order to clone them into the digested pet24d vecor.

The restriction was performed for 1h at 37°C (heating block).

After the plasmid and the two inserts have been digested, two ligations and a ligation control were performed in order to gain the final expression vectors.

For the optimal ligation results the ligtion calculator of the iGEM team Dallas was used. Ligation was performed for 2h. Afterwards the ligase was inactivated at 65°C for 15 min in order to improve the ligation/transformation result.

After the ligase inactivation the whole reaction mix was transformed into E.coli XL-blue which seem to be the best cells for important transformations. Standard transformation protocol was used.

The Gibson assembly plate showed sixteen colonies. All colonies were picked, transferred onto a master plate and into a premixed colony PCR. The same colonieswereusedtoinoculate a miniprep.

The colony PCR was negative, even the positive control with the amplified KS part I with the flanks.

The plasmids were prepped and digested with KpnI and SacI to test the insertion of the KS part I into pMAD. The expected sizes were 5054 bp and 6402 bp with the insert and pMAD only would yield in 4440 bp, 170 bp and 5054 bp fragments.

The clones1, 2, 5, 7, 8, 9, 10, 11, 12 and 14 showed the expected results. The rest of the isolated plasmids were discarded.

The overnight cultures from 18.70 were harvested for 10 min at 4000 x g and the pellets were frozen away at -80°C until use.

A preculture of 10 ml LB was inoculated with B. subtilis WT3610 cells and incubated at 37°C over night.

The midi prep of the plasmids piGEM002 with the silver and copper promoters and the killswitch promoters was carried out according to the Qiagenmidiprep kit instructions. The prepped plasmids were tested via restriction with BamHI and SpeI.

Precultures of all four strains (JH642, PY79, 168 and WT3610) were inoculated in 3 ml HS-medium and 10 ml LB and were incubated over night at 37°C.

After the successful ligation (colonies in both plates), 5 clones per ligation have been inoculated in LB-Kan Medium for a later Mini-prep and test restriction of the expression plasmids. Colonies with the numbers 6-10 were chosen as 1-5 did not grow over the day. 6-10 were incubated over night.

The only preculture, which was grown in HS was the JH642. Every other strain did not grow in High Salt medium. 20 ml of prewarmed (30°C) Low Salt medium were inoculated with 1 ml of the HS culture and incubated in a shaking waterbath at 30°C with 120 rpm for 3 hours. 5 µg of the plasmids were preset in Eppendorf cups and 1 ml of the LS culture was transferred into these cups. piGEM002 was transformed as a control. These culture/plasmid mixes were incubated at 37°C shaking at 200 rpm. After 2 hours of incubation the cells were centrifuged at 8000 rpm for 1.5 minutes, most of the medium was withdrawn,the cells were resuspended in the rest of the medium and plated out on LB-Cm plates that were incubated at 37°C over night.Precultures of all strains were again inoculated, since many were not grown.

The precultures in LB medium were all grown. 10 ml Spizizens minimal medium were inoculated with an OD600 of 0.1. The cultures wre incubated at 37°C until they reached an OD of 1. 5 µg of DNA were mixed with 1 ml of the culture and incubated for further 2 hours. 200 and 500 µl of each transformation sample were plated out on separate LB-Cm plates, which were incubated at 37°C over night.

10 ml of MNGE-medium were inoculated 1/100 with the preculture. This culture was grown at 37°C with an agitation of 200 rpm until it reached OD 1.4. 5 µg of he prepped plasmids pMAD-KSI from clone 1, 2, 10 and 11 (22.12) were used to perform four transformations. 400 µl of the cells were added to the plasmids in a test tube and were incubated at 37°C 200 rpm for 1 hour. 100 µl expression mix were added and the cells were incubated for another hour. Dilutions up to 10-6 were carried out and the 10-5 and 10-6 dilutions were plated out on LB-MLS-X-Gal plates that were incubated at 30°C for several days.

Plasmids were extracted from the cells and a test restriction was performed with 300 ng plasmid. Gelfoto was unfortunately not saved but the bands were at the expected size (appr. 1500 bp). Therefore one plasmid each was sent for sequencing. Cup Number 6!